This invention relates to a method and means of genetic control of ethylene biosynthesis in plants.
Ethylene is a plant hormone influencing many aspects of plant growth and development. This simplest of all unsaturated carbon compounds is a powerful regulator of plant metabolism, acting, and interacting with other plant hormones in trace amounts.
Ethylene promotes senescence in plants, both in selected groups of cells and in whole organs such as fruits, leaves, or flowers. Senescence is the natural, genetically controlled degenerative process which usually leads to death in plants. Even at low concentrations (ethylene is a gas under physiological conditions), ethylene has profound hormonal effects on plants. The effects of ethylene, whether produced by the plant itself or applied exogenously, are numerous, dramatic, and of considerable commercial importance. Among the diverse physiological effects are:
a. Stimulation of ripening in fruits and vegetables PA1 b. Leaf abscission PA1 c. Fading in flowers PA1 d. Flower wilting PA1 e. Leaf yellowing PA1 f. Leaf epinasty
Normally, ethylene production from plant tissue is low. Large quantities of ethylene, however, are produced during ripening and senescence processes. A large amount of ethylene is also produced following trauma caused by chemicals, temperature extremes, water stress, ultraviolet light, insect damage, disease, or mechanical wounding. Ethylene produced by plants under such conditions is referred to as "wound ethylene" or "stress ethylene". In fruits and vegetables, the stimulation of ethylene production by cuts or bruises may be very large and bear considerably on storage effectiveness. Ethylene-induced leaf browning is a common basis for loss in many plants, including lettuce and tobacco. In some tissues, exposure to only a small amount of ethylene may cause an avalanche of ethylene production in adjacent plants or plant tissues such as fresh produce. This autocatalytic effect can be very pronounced and lead to loss of fruit quality during transportation and storage.
The mechanism by which ethylene exerts its effects has become apparent only in the last few years. As judged by numerous data, each of the responses to ethylene involves an ethylene receptor site--a metalloenzyme. The reaction of ethylene with its receptors triggers a cascade of physiological events. Marked increases in the amounts of RNA and protein occur in response to ethylene. The levels of several enzymes have also been shown to increase in response to ethylene, such as cellulase, .alpha.-amylase, and invertase.
Current technologies that specifically address post-harvest storage life have been in existence for decades and are hampered by such problems as high cost, side effects, and an inability to completely shut off ethylene production. Included in this group are controlled atmosphere (CA) storage, chemical treatment, packaging, and irradiation.
CA facilities slow ethylene biosynthesis through: (1) low temperature, (2) reducing the oxygen level below 3%, and (3) elevating the carbon dioxide level in the storage area to the 3%-5% range. Expensive scrubbers are sometimes added which reduce ethylene already respired to the atmosphere. Drawbacks are that CA facilities are expensive to construct, have a high utility cost, and are unable to completely eliminate ethylene production and side effects. Also, CA storage techniques can only control external ethylene and not that which resides inside the plant tissue. CA storage can also lead to undesirable side effects. Injury can result from high CO.sub.2 levels, low O.sub.2 levels, or low temperature.
Another approach is to limit ethylene biosynthesis in the plant tissue through chemical treatment. Aminoethoxyvinylglycine (AVG), an analog of the antibiotic rhizobitoxine, is such an inhibitor. Use of the chemical in foods is impossible, however, due to its high toxicity. Silver thiosulfate (STS) is also effective in slowing fruit ripening and flower fading but is also toxic and cannot be used on foods. STS only works with certain flowers and often causes black spotting.
The amino acid methionine has been shown to be a precursor of ethylene in plant tissues. Methionine, however, is not the immediate precursor, but first must be converted to the sulfonium compound S-adenosylmethionine (AdoMet) and, subsequently to 1-aminocyclopropane-1 carboxylic acid (ACC) prior to conversion to ethylene. The following metabolic reactions (also see FIG. 1) are now accepted for the synthesis of ethylene from methionine under both normal and stress conditions: EQU Methionine.fwdarw.AdoMet.fwdarw.ACC.fwdarw.Ethylene
The system which converts ACC to ethylene appears to be constitutive in most plant tissues with the notable exception of some preclimacteric fruit tissue. ACC synthase catalyzes the degradation of AdoMet to ACC and 5'-methylthioadenosine (MTA). This enzymatic reaction seems to be the rate limiting step in ethylene formation. AdoMet is synthesized via a condensation reaction between methionine and Adenosinetriphosphate (ATP). Attempts at regulating the levels of AdoMet by controlling the rate of AdoMet synthesis have failed, mainly because there appear to be at least three different AdoMet synthesizing enzymes coded by three different genes. In addition, the known biochemical inhibitors of AdoMet synthesis are very toxic to mammalian cells. See S. F. Yang, et al., "Ethylene Biosynthesis and its Regulation in Higher Plants," Ann. Rev. Plant Physiol, 35:155-189, 1984; Veen, et al., SciHortic, 18:277-286; Sisler, et al, Plant Physiol, 63:114-120; and Wang, et al., Plant Physiol, 89:434-438.
Although plant tissues are known to maintain a substantial rate of ethylene production for extended periods, their methionine levels have been shown to be very low. To continue to produce ethylene, the sulfur contained in MTA must be recycled back into methionine so as to provide an adequate supply of methionine for continual ethylene production. This pathway has been recently shown to exist in plant tissue (see FIG. 1c). See also S. F. Yang, et al., "Ethylene Biosynthesis and its Regulation in Higher Plants," Ann. Rev. Plant Physiol, 35:155-189, 1984. The degradation of MTA has added significance in light of the finding that MTA is a potent inhibitor of ACC synthase. It should be noted that this pathway merely maintains a methionine supply for ethylene biosynthesis, but does not result in a net increase in methionine synthesis.
An enzyme encoded by the E. Coli bacteriophage T3 hydrolyzes S-adenosylmethionine (AdoMet) to homoserine and 5'-methylthioadenosine (MTA). This enzyme is known by either its recommended name, AdoMet hydrolase (AdoMetase), or by its other name, S-adenosylmethionine cleaving enzyme (SAMase). See Studier, et al., "SAMase Gene of Bacteriophage T3 is Responsible for Overcoming Host Restriction," Journal of Virology, 19:135-145, 1976. Both products of the reaction are recycled to methionine; MTA as previously shown (FIG. 1) and homoserine via a metabolism pathway known to exist in plant tissues. The AdoMetase gene has been identified, isolated, cloned, and sequenced. J. A. Hughes, et al., "Expression of the Cloned Coliphage T3 S-adenosylmethionine Gene Inhibits DNA Methylation and Poly Amine Biosynthesis in Escherichia coli", J. Bact., 169:3625-3632, 1987 and J. A. Hughes, et al., "Nucleotide Sequence and Analysis of the Coliphage T3 S-adenosylmethionine Hydrolase Gene and its Surrounding Ribonuclease III Processing Sites", Nuc. Acids Res., 15:717-729, 1987. The gene contains two inframe reading sequences that specify polypeptides of 17105 and 13978 daltons. Both polypeptides terminate at the same ochre codon. This results in the 14 kd polypeptide being identical to 82% of the 17 kd polypeptide starting at the carboxyl end of the longer polypeptide. Both polypeptides are present in partially purified preparations of active AdoMetase from T3 bacteriophage infected cells and from E. Coli expressing the cloned gene. J. A. Hughes, et al., "Nucleotide Sequence and Analysis of the Coliphage T3 S-adenosylmethionine Hydrolase Gene and its Surrounding Ribonuclease III Processing Sites," Nuc. Acids Res., 15:717-729, 1987 and F. W. Studier, et al., "SAMase Gene of Bacteriophage T3 is Responsible for Overcoming Host Restriction ," J. Virol., 19:136-145, 1976.
Other bacteriophages that encode the AdoMetase or SAMase genes are coliphage BA14, Klebsiella phage K11, and Serratia phage IV. See H. Mertens, et al., "Coliphage BA14: a New Relative of Phage T7," J. Gen. Virol., 62:331-341, 1982; R. Hausmann, The Bacteriophages, 1:279-283, 1988, R. Calender (ed.), Plenum Press, New York; and K. H. Korsten, et al., "The Strategy of Infection as a Criterion for Phylogenetic Relationships of Non-Coli Phages Morphologically Similar to Phage T7," J. Gen. Virol., 43:57-73, 1979.